Determination of vitamins A, D and E in a small volume of human plasma by a high‐throughput method based on liquid chromatography/tandem mass spectrometry

We have developed an automated high‐throughput assay for the determination of vitamin A (retinol), ergocalciferol (25‐OH D2), cholecalciferol (25‐OH D3) and vitamin E (α‐tocopherol) in a small volume of human plasma. Sample preparation involved mixing 50 μL of plasma with 100 μL of ethanol containing isotope‐labelled internal standards, followed by mixing with isooctane/chloroform (3:1, 300 μL). The organic phase was evaporated, and the sample reconstituted in 50 μL methanol. The analysis was performed using reversed‐phase liquid chromatography with a gradient mobile phase containing water, methanol and ammonium formate. Chromatographic run‐time was 5 min, and positive mode electrospray tandem mass spectrometry (MS/MS) was used for detection. The limits of detection were 0.10 μM for all‐trans retinol and 3.3 nM for 25‐OH D2 and 25‐OH D3. Recoveries were 91.9–105.0%, and within‐ and between‐day coefficients of variance (CVs) 2.4–5.3 and 3.1–8.2, respectively. The assay is presently being used in large‐scale studies. Copyright © 2011 John Wiley & Sons, Ltd.